Mutagens and carcinogens in size-classified air particulates of a Northern Italian town

The Science of the Total Environment 205 (1997) 137-144 ELSEVIER

Mutagens and carcinogens in size-classified air particulates of a Northern Italian town

S. Monarcaaj* , R. Crebellib, D. Feretti”, A. Zanardini”, S. Fusellib, L. Filinid, S. Resolad, P.G. Bonardellid, G. Nardi”

“Chair of Hygiene and Preventive Odontoiatry, Medicine Faculty, University of Brescia, Via Vakabbina 19, I-25123 Brescia, Italy

blstituto Superiore di Sank?, Rome, Italy ‘Chair of Hygiene, Medicine Faculty, University of Brescia, Brescia, Italy

‘PMIP, Local Health Unit No. 18, Brescia, Italy

Received 19 February 1997; accepted 25 June 1997

Abstract

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulate matter in relation to particles aerodynamic size. Inhalable (< 10 pm) airborne particulate (PM-101 was collected at a low traffic site in an industrialized Northern Italian town, using a high volume sampler equipped with a cascade impactor for particles fractionation. The organic extracts of different fractions were examined for mutagenicity in Salmonella typhinwium strains TA98 and TA98/1,8-DNP, using the microsuspension procedure, and for polycyclic aromatic hydrocarbons (PAHs) content by gas chromatography. Size fractionated particles were also analysed for heavy metals (Fe, Mn, Zn, Pb, Cu, Cd, Cr, Ni, V) using plasma spectrophotometry. The results of mutagenicity and chemical analyses indicate that, at the site investigated, inhalable particulate was largely made of fine (< OSpm) particles, which accounted for most of PAHs and mutagenicity. A similar pattern of distribution was found for heavy metals, which were relatively more abundant in small ( < 1.5 pm> particles compared to coarser ones. 0 1997 Elsevier Science B.V.

Keywords: Airborne particulate; Size-classified particulate; Mutagens; Carcinogens; PAH; Heavy metals

* Corresponding author. Tel.: + 39 30 3994402; fax: + 39 30 3701404; e-mail: [email protected]

004%9697/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIZ SOO48-9697(97) 00194-o

138 S. Monarca et al. /The Science of the Total Environment 205 (1997) 137-144

1. Introduction

Research has shown that urban air, particularly in industrialized areas, contains a variety of known mutagenic and carcinogenic substances, including organic compounds such as benzo[a]pyrene and benzene, inorganic compounds such as nickel and chromium and radionuclides (Fishbein, 1993; Lewtas, 1993). Experimental studies have shown that extracts of urban air particles can induce skin cancer in animals (Hueper et al., 1962; Ep- stein et al., 1979; Sasaki et al., 1987) and are mutagenic in bacteria (Kado et al., 1986; Matsu- moto and Inoue, 1987; Crebelli et al., 1988, 1995; De Raat, 1988; Viras et al., 1990, 1991; Fuselli et al., 1995; Pagan0 et al., 1996) and in mammalian cells (Krishna et al., 1984; Hadnagy and See- mayer, 1986; Hadnagy et al., 1989). Moreover, several epidemiological studies carried out over the last 40 years to investigate the health risk related to air pollution suggested a possible increased lung cancer risk for urban populations (Schwartz, 1991; Summerhays, 1991; Tzonou et al., 1992; Pershagen and Simonato, 1993). There- fore the monitoring of urban air for genotoxic micropollutants, in addition to conventional toxic pollutants (e.g. SO,, NO,, CO), is receiving increasing consideration to the aim of evaluating health risks for people living in urban areas.

using both bacterial mutation assays and chemical methods. Emphasis was given to the analysis of the distribution of both mutagens and organic (PAHs) and inorganic (heavy metals) carcinogens in air particulate in relation to particle aerodynamic size, in view of the greater ability of smaller particles to reach the bronchial and alveolar por- tions of the respiratory tract.

2. Material and methods

2.1. Air samplings

Brescia is the second largest town in Lombardy (approx. 200 000 inhabitants; 100 km from Milan). The town is heavily industrialized, featuring mainly foundries and metal industries. From the beginning of the 197Os, air pollution emissions from diffuse heating sources were reduced because of the adoption of a district heating system, the largest in Italy, used by more than 50% of the population.

Air pollution is presently considered a primary health problem in Lombardy, a highly industrialized and urbanized region of North Italy, with lung cancer rates higher than the Italian average. In this region, ambient air quality standards for regulated air pollutants are sometimes exceeded, with consequent endorsement of temporary emer- gency measures such as traffic restriction in urban areas or prohibition for circulation of uncatalyzed vehicles. However, few if any information is avail- able on local levels of airborne mutagenic/ carcinogenic pollutants, especially in the finest, deeply inhalable particulate matter.

Samples of inhalable (< 10 pm) airborne particulate matter (PM-lo) were collected during the spring of 1991 in a residential area of the town, with moderate car traffic. Particles were collected on preweighted glass fiber filters using a high volume sampler (High Volume PM-10 sampler, Andersen) placed at the street level. In one sampling (A) the total PM-10 was collected, whereas in other samplings (B, C, D and E) a 5-stage cascade impactor (Andersen) was applied down- stream of the high-volume sampler, in order to separate particles according to their aerodynamic size in five classes: 7.2-10 pm (stage l), 3-7.2 pm (stage 2), 1.5-3 pm (stage 3), 0.95-1.5 pm (stage 4), 0.5-0.95 pm (stage 5) and < 0.5 pm (final stage). Filters were stored at -20°C in the dark until sample preparation.

2.2. Sample preparation

The aim of this work was to study the content Glass-fiber filters were dried at room tempera- of mutagenic and carcinogenic compounds in air ture, weighted and extracted for 300 cycles in a particles. collected at a typical medium-size Lom- Soxhlet apparatus with 200 ml of dichloromethane bard town, characterized by high industrial pollu- (DCM). In order to obtain sufficient amount of tion and a relatively moderate traffic intensity, extracts for mutagenicity tests and PAH analysis,

S. Monarca et al. /The Science of the Total Environment 205 (1997) 137-144 139

filters from samplings B, C and D were pooled together according to the granulometry of particles collected and their extracts combined in two larger samples: 0.5-10 pm (stages l-5) and < 0.5 pm (final stage). The extracts were evaporated to dryness with a rotating evaporator and the residues stored in the dark at -20°C until analyses for mutagenicity and PAH content. Filters of sampling E were used for metal analysis with no previous extraction.

2.3. Airborne particulate mutagenicity analysis

The mutagenicity of air particulate extracts was analysed in the so-called Kado test, a simple modification of the SalmonelZa/microsome liquid-incubation procedure (Kado et al., 1983). This procedure was previously found to be especially sensitive to airborne mutagens (Kado et al., 1986; Lewtas et al., 1990; Bagley et al., 1992) and it was recommended for the analysis of complex environmental mixtures (Agurell and Stensman, 1992; Claxton et al., 1992). Briefly, air particle extracts were dissolved in dimethyl sulfoxide (DMSO) and analyzed in triplicate plates at three increasing concentrations (50, 100 and 200 pg/plate), with and without exogenous metabolic activation (pro- vided by liver S9 from Aroclor pretreated rats), using the S. typhimurium tester strains TA98 and TA98/1,8-DNP,. The latter strain is deficient in O-ace@ transferase and resistant to the mutagenicity of nitropyrenes (McCoy et al., 1983) and it is widely used as an indicator of the presence of nitroarenes in complex mixtures. Positive control substances were 4-nitro-o-phenylenediamine for TA98 - S9, 4-nitroquinoline-l-oxide for TA98/1,8-DNP, - S9, 2-acetylaminofluorene for TA98 + S9 and benzo[a]pyrene for TA98/1,8- DNP, + S9. Pure DMSO and extracts of filter blanks were also tested as negative controls.

The results of mutagenicity assays are ex- pressed as specific mutagenic activity, i.e. induced his+ revertants/mg of tested material, calculated by linear regression analysis of the linear part of the dose-response curves. These values were also used to calculate air mutagenicity levels (i.e. induced revertants/m3) on the basis of the content of organic extract/m3.

2.4. Chemical analysis

The PAH concentrations were determined by high-resolution gas chromatography (HRGC) on extracts of the pooled samples B, C and D after a purification process of the samples themselves.

The analysis was carried out using a Hewlett- Packard mod. 5990 gas chromatograph. The oper- ating conditions were the following:

l Column: SE-54 (25 m X 0.32 mm X 0.52 pm> . Detector: FID l Detector temperature: 300°C 0 Injection system: on-column . Carrier gas: helium . Column temperature:

1. 80°C (initial temperature) 2. 30”C/min increase up to 120°C 3. 4”C/min increase up 280°C (final tempera-

ture).

The detection limit was 0.1 ng/m3 for each compounds. The PAH contents were calculated on the basis of recovery tests carried out with a standard mixture.

Recovery percentages, tested in several experiments, were close to 100%. A somewhat lower percentage was, however, detected in the case of low molecular weight compounds. The reference PAH mixture EPA 610 (Supelco Inc., USA) was used as standard.

Filters of the sampling E were analyzed for the content of heavy metals (Fe, Mn, Zn, Pb, Cu, Cd, Cr, Ni, V) by ICP-AES analysis with plasma spectrophotometry JY24 (Jobin-Yvon Instruments, Longjumeau, France). The filters were treated with nitric acid to oxidize the organic matrix and to dissolve the metals. Blanks were also per- formed. The detection limits were 5.3, 7.2, 6.3, 2.6, 2.8, 23.8 and 3.2 rig/ml for Zn, Pb, Fe, Mn, Cr, Cu and Ni respectively. Multielement solution (MSQ-6, C. Erba, Milan, Italy) was used as standard.

3. Results

Table 1 shows weight values and content of


Table 1 Samplings of airborne particulate matter in the city of Brescia (weight values and gaseous pollutant levels)

Sample (date)

Filtered Particulate collected (mg) Particles Organic Organic air Total PM-10 o,5-10 < o.5 (&m3) emact

w pm (4 (~4

A (27 March 1991) 1545 105.8 68.5 12.7 8.2 70 32 B (19 March 1991) 1483 144.4 53.2 91.2 97.4 78 31 C (26 March 1991) 1545 54.1 18.3 35.8 35.0 70 33 D (9 April 1991) 1450 185.0 37.8 147.2 127.6 56 18 B-D (pooled) 4478 29.0* 6.5*

36.0** 8.1** E (29 November 1991) 1484 193.2 - 130.2 - 41 14

* 0.5-10 Km. ** <0.5 Km.

dichloromethane extractable material in airborne particles sampled at a residential site in Brescia. The total concentration of inhalable particles (total PM-101 were in most cases above the Ital- ian air quality standard (60 pg/m3>, and in one case (sample D) also above the recommended US standard (150 pg/m3 as daily mean). The content of organic matter from the unfractionated air

particles sample (12% by weight, sample A> was fairly similar to the figures reported from previous studies in other Italian cities (Crebelli et al., 1995; Fuselli et al., 1995). On the other hand, a larger amount of organic extract (13.3-26.4%) was obtained from the pooled filters of samplings B-D, possibly in connection to different meteorological conditions. Interestingly, in these samples

” ’ c 0.5 ’ 0.5-0.95 I 0.95-1.5 I 1.5-3 3-7.2

PARTICLE SIZE (urn)

Fig. 1. Distribution of PM-10 particulates collected during samplings B, C and D in Brescia and fractionated according to the aerodynamic size using a 5-stage cascade impactor.

S. Monarca et al. / The Science of the Total Environment 205 (1997) 137-144 141

a relatively greater amount of organic matter was obtained from finest particles, with aerodynamic diameter < 0.5 pm.

In the same table, the average levels of SO, and NO, recorded during samplings are shown: both gaseous pollutants were below the Italian air quality standards (125 pg/m3 for SO,, mean of 24 h and 200 pg/m3 for NO,, mean of 1 h) during particles collection.

Fig. 1 shows size distribution of airborne particles fractionated by granulometry using the An- dersen 5-stage cascade impactor (samples B-D). The major contribution to the total PM-10 matter (63-80%) was given by the finest fraction (< 0.5 pm), whereas larger particles contributes to a more limited extent.

Table 2 summarizes the results of mutagenicity assays with both non-fractionated and fraction-

ated airborne particulates. All samples were found to be mutagenic in strain TA98 in the absence of exogenous metabolic activation, revealing the presence of direct-acting mutagens. The addition of S9 mix did not significantly increase mutagenic activities. In the fractionated samples, the extract of fine particles ( < 0.5 pm) was more mutagenic, on a weight basis, than the extract of coarser particles (0.5-10 pm). From the above data, and from the weight values shown in Table 1, air mutagenicity levels (induced revertants/m3) are calculated, which indicate that finest particles give the prevalent contribution to air mutagenicity.

Parallel experiments with strain TA98/1,8- DNP, gave mainly inconclusiveresults with the unfractionated sample, because of the high toxicity elicited by the sample in this strain, especially in experiments without S9. The lower viability of

Table 2 Mutagenicity of airborne particulate matter extracts in Salmonella typhimutium tester strains (results from microsuspension test experiments)

Sample

A (unfractionated) B-D (0.5-10 pm) B-D (< 0.5 pm)

* Toxic effect.

Induced revertants/mg of extract

TA98 TA98 TA98/ -s9 +s9 1,8-DNPG

-s9

520 577 o.o* 310 369 263 942 697 860

TA98/ 1,8-DNP, +s9

116* 313 422

Induced revertants/m3 of air

TA98 TA98 TA98/ -s9 +s9 1,8-DNP,

-s9

4.3 4.1 0.0 2.0 2.4 1.7 7.6 5.6 7.0

TA98/ 1,8-DNP, +s9

0.9 2.0 3.4

Table 3 Polycyclic aromatic hydrocarbon (PAH) concentration in samples of airborne particulate matter sampled in the city of Brescia

Sample

A (unfractionated) B-D (0.5-10 pm) B-D (< 0.5 pm)

PAH* ( pg/mg of extract)

BaA Chr +Tri Pe BaP BeP BghiPe Py XPAH

0.46 1.15 0.13 0.35 0.96 1.77 5.26 10.08 0.04 0.03 0.02 0.02 0.04 0.05 0.26 0.46 0.25 0.29 0.07 0.22 0.42 0.53 2.09 3.87

PAH* (ng/m3 of air)

A (unfractionated) 3.77 9.41 1.07 2.82 7.83 14.47 42.87 82.24 B-D (0.5-10 pm) 0.25 0.22 0.15 0.16 0.27 0.30 1.72 3.07 B-D (< 0.5 pm) 2.04 2.36 0.58 1.78 3.43 4.34 16.96 31.49

* PAH: polycyclic aromatic hydrocarbon; BaA, benzo[a]anthracene; Chr + Tri, chrysene + triphenylene; Pe, perylene; BaP, benzo[a]pyrene; BeP, benzo[e]pyrene; BghiPe, benzo[ghi]perylene; Py, pyrene.


this TA98 derivative, compared to the parent strain, may explain the greater toxicity elicited by the test material in this strain. On the other hand, the results obtained with organic extracts of fractionated particles were basically comparable in the two strains (Table 2, samples B-D), suggesting a negligible contribution of nitroarenes and other nitrogen containing compounds to the mutagenicity of this sample.

Table 3 shows the concentrations of PAH in fractionated and non-fractionated particulate matter. Trace amounts of carcinogenic PAH (benzo[ alpyrene, benzo[ alanthracene) were found in all samples. The comparison of PAH concentrations in particles of different size shows far larger concentrations of all PAHs identified in finest particles (< 0.5 pm>, which alone accounted for approx. 90% of the total PAH content in samples B-D.

The concentrations of carcinogenic and non- carcinogenic heavy metals was measured in another fractionated air particles sample (sample E), which was processed for analysis without previous extraction. Data in Table 4 show heavy metal concentrations in sample E which are comparable to the levels usually found in other Euro- pean industrialized areas (World Health Organi-

zation, 19871, possibly reflecting the proximity to foundries and metal industries to the sampling site. Similarly to what observed for organic pollutants, also in this case the greater concentrations of heavy metals were found in small size particles (< 1.5 pm).

4. Discussion

This study was aimed at evaluating the presence of organic and inorganic mutagens and carcinogens in airborne particulate collected at a typical industrialized Northern Italian town. The sampling site was located in a residential area, where neighbouring industrial activities are sup- posed to be a major source of atmospheric pollution, even though the contribution of automobile exhausts cannot be dismissed. Mutagenicity test- ing of dichloromethane extracts of particulate matter, using a very sensitive bacterial assay, showed in all samples the presence of direct-acting mutagens, including species different from ni- troaromatics. A similar activity profile was previously reported in studies on the organic extract of airborne particulates collected in another urban area (Crebelli et al., 1988). The fractionation of particles by size, using a cascade impactor, de-

Table 4 Heavy metals concentrations in size-fractioned airborne particles collected in the city of Brescia

Particle size ( pm)

10.0-7.2

7.2-3.0

3.0-1.5

1.5-0.95

0.95-0.5

< 0.5

Total PM-10

Concentration of heavy metals in air particles

pg/m3 (% distribution by particle size)

Fe Mn Zn Pb

0.93 0.04 0.03 0.08 (19.3) (2.8) (0.7) (4.2)

0.67 0.58 0.70 0.12 (13.9) (40.6) (15.3) (6.3

0.03 0.03 0.28 0.09

(0.6) (2.1) (6.1) (4.7) 2.15 2.58 1.06

(44.5) (3::: (56.3) (55.5) 0.51 0.20 0.65 0.23

(10.6) (14.0) (14.2) (12.0) 0.54 0.12 8.34 0.33

(11.2) (8.4) (7.4) (17.3) 4.83 1.43 4.58 1.91

(100.0) (100.0) (100.0) (100.0)

“g/m3 (% distribution by particle size)

cu Cd Cr Ni

0.05 17.3 (10.9) (2) (12) (16.7)

0.04 4.4 11.7 (8.7) (llf, (11.9) (11.3) 0.02 24.0

(4.3) (E) (1:::) (23.1) 0.20 13.2 23.0

(43.5) (3;::) (35.7) (22.1) 0.09 11.3

(19.6) (2::;) (2, (10.9) 0.06 16.6

(13.0) (l$ (2;::) (16.0) 0.46 30.4 37.0 103.9

(100.0) (100.0) (100.0) (100.0)

V

6.9 (10.8) 11.7

(18.3) 5.3 (8.3) 13.2

(20.7) 14.2

(22.3) 12.5

(19.6) 63.8

(100.0)

S. Monarca et al. /The Science of the Total Environment 205 &?97) 137-144 143

monstrated that suspended material was mainly composed of very fine particles (< 0.5 pm). Mu- tagenicity assays with fractionated samples demonstrated that the organic extract of finest particles was the most mutagenic, also on a weight basis, suggesting a different qualitative composi- tion of the organic matter absorbed on airborne particles in relation to particle size, possibly related to their different deposition rates and atmospheric behaviour. This is conceivable in view of the well known extensive atmospheric transfor- mation of precursor mutagenic compounds (Al- brecheinski et al., 1985; Takeda and Teranishi, 1986).

A greater contribution to total air mutagenicity by finest particles was also reported in studies carried out in Greece (Viras et al., 1990, 1991), the Netherlands (De Raat, 1988) and Italy (Pagan0 et al., 1996), thus indicating that this phenomenon may be a general feature of the distribution of genotoxins in airborne particles. In addition, in this study a similar patter of distribution was also demonstrated for several carcinogenic and non-carcinogenic PAHs and heavy metals. Similar results were reported very recently in an independent work (Schnelle et al., 1996). Obvi- ously PAH, which are proximate mutagens, do not play themselves a significant role in the response elicited by airborne particle extracts, which are typically direct acting mutagens. Rather, PAHs can be regarded as tracers of related compounds (mainly oxygenated derivatives) which play a major role in air particles mutagenicity in bacteria (Matsumoto and Inoue, 1987).

In conclusion, the results of this work suggest that the potential cancer risk associated to air particulate pollution is mainly related to the in- halation of fine particles (< 1.5 pm), which con- tain the highest amount of both organic and inorganic mutagens/carcinogens. These particles have greater possibilities to exert a detrimental effect, reaching the deep respiratory tract and lung alveoli. These conclusions are in line with the recently proposed US air quality standard for fine particles, which considers PM-2.5 instead of PM-10 as a more significant parameter for the evaluation of health hazards related to air pollution (Reichhardt, 1996).

Acknowledgements

The authors are grateful to Mrs B. Crochi, A.R. Proietto and Mr L. Conti for technical assis- tance. This work was partially supported by Azienda Servizi Municipalizzati, Public Utilities, Brescia and by MURST grant (60% funds).

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Mutagens and carcinogens in size-classified air particulates of a Northern Italian town